Multitag sequencing ecogenomics analysis

ABSTRACT

Embodiments of the invention herein described relate to multiplex polynucleotide sequence analysis without the use of size separation methods or blotting. In certain particulars the invention relates to multiplex sequencing using massively parallel sequencing methods, such as pyrosequencing methods and sequencing by synthesis. The invention provides increased throughput, increased accuracy of enumerating sample components, and the ability to analyze greater numbers of samples simultaneously or serially on presently available systems, as well as others yet to be developed. In certain of its embodiments the invention relates to the analysis of complex microbial communities, particularly to in-depth analysis thereof in large numbers of samples.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/515,262filed May 15, 2009 (now U.S. Pat. No. 8,603,749), which is a NationalStage of PCT/US07/84840, filed Nov. 15, 2007, which claims full benefitof priority of U.S. provisional application No. 60/858,948, filed on 15Nov. 2006, each of which is hereby incorporated by reference in itsentirety.

STATEMENT OF GOVERNMENT RIGHTS

Work described herein was done partly with Government support underGrant No. 1R43DK074275-01A2 awarded by the U.S. National Institute ofDiabetes and Digestive and Kidney Diseases, and the US Governmenttherefore may have certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to the determination of polynucleotide sequences.It also relates to determining sequences in multiple samples, in someparticulars in multiple environmental samples and in multiple clinicalsamples.

BACKGROUND

Sequence determination technologies for proteins, RNAs and DNAs, havebeen pivotal in the development of modern molecular biology. During thepast fifteen years, DNA sequencing in particular has been the coretechnology in an on-going revolution in the scope and the depth ofunderstanding of genomic organization and function. The on-goingdevelopment of sequencing technology is, perhaps, best symbolized by thedetermination of the complete sequence of a human genome.

The human genome sequencing project served a number of purposes. Itserved as a platform for programmatic development of improved sequencingtechnologies and of genome sequencing efforts. It also served to providea framework for the production and distribution of sequencinginformation from increasingly large scale sequencing projects. Theseprojects provided complete genome sequences for a succession of modelorganisms of increasingly large genetic complements. Theseaccomplishments, culminating in the completion of a human genomesequence, highlight the very considerable power and throughput ofcontemporary sequencing technology.

At the same time, however, they highlight the limitations of currenttechnology and the need for considerable improvements in speed,accuracy, and cost before sequencing can be fully exploited in researchand medicine. Among the areas that can be seen most readily to requireadvances in sequencing technology are clinical sequencing applicationsthat require whole genome information, environmental applicationsinvolving multiple organisms in mixtures, and applications that requireprocessing of many samples. These are, of course, just a few among agreat many areas that either require or will benefit greatly from morecapable and less expensive sequencing methods.

To date, virtually all sequencing has been done by Sanger chainelongation methods. All Sanger methods require separating the elongationproducts with single base resolution. Currently, while PAGE still isused for this purpose in some commercial sequencers, capillaryelectrophoresis is the method of choice for high throughput DNAsequencers. Both gel-based and capillary-based separation methods aretime consuming, costly, and limit throughput. Chip based methods, suchas Affymetrix GeneChips and HySeq's sequencing by hybridization methods,require chips that can be produced only by capital intensive and complexmanufacturing processes. These limitations pose obstacles to theutilization of sequencing for many purposes, such as those describedabove. Partly to overcome the limitations imposed by the necessity forpowerful separation techniques in chain termination sequencing methodsand the manufacturing requirements of chip-based methods, a number oftechnologies are currently being developed that do not require theseparation of elongation products with integer resolution and do notrequire chips.

A lead technology of this type is a bead, emulsion amplification, andpyrosequencing-based method developed by 454 Life Sciences. (SeeMarguilles, et al. (2005) Nature 437: 376, which is incorporated hereinby reference in its entirety, particularly as to the aforementionedmethods. The method utilizes a series of steps to deposit single,amplified DNA molecules in individual wells of a plate containingseveral million picoliter wells. The steps ensure that each well of theplate either contains no DNA or the amplified DNA from a single originalmolecule. Pyrosequencing is carried out in the wells by elongation of aprimer template in much the same way as Sanger sequencing.Pyrosequencing does not involve chain termination and does not requireseparation of elongation products. Instead sequencing proceeds stepwiseby single base addition cycles. In each cycle one of the four bases—A,T, G, or C—is included in the elongation reaction. The other three basesare omitted. A base is added to the growing chain if it is complementaryto the nest position on the template. Light is produced whenever a baseis incorporated into the growing complimentary sequence. Byinterrogating with each of A, C, G, or T in succession, the identity ofthe base at each position can be determined. Sequencing reactions arecarried out in many wells simultaneously. Signals are collected from allthe wells at once using an imaging detector. Thus, a multitude ofsequences can be determined at the same time.

In principle, each well containing a DNA will emit a signal for only oneof the four bases for each position. In practice, runs of the same baseat two or more positions in succession lead to the emission ofproportionally stronger signals for the first position in the run.Consequently, reading out the sequence from a given well is a bit morecomplicated then simply noting, for each position, which of the fourbases is added. Nevertheless, because signals are proportional to thenumber of incorporations, sequences can be accurately reconstructed fromthe signal strength for most runs.

The technology has been shown to read accurately an average of about 250or so bases per well with acceptable accuracy. A device offered by 454Life Sciences currently uses a 6.4 cm² picoliter well “plate” containing1,600,000 picoliter sized wells for sequencing about 400,000 differenttemplates. The throughput for a single run using this plate currently isabout 100 million bases in four hours. Even though this is a firstgeneration device, its throughput is nearly 100 times better thanstandard Sanger sequencing devices.

Numerous other methods are being developed for ultra high throughputsequencing by other institutions and companies. Sequencing by synthesismethods that rely on target amplification are being developed and forcommercialized by George Church at Harvard University, by Solexa, and byothers. Ligation sequencing methods have been developed and/or are beingcommercialized by Applied Biosystems and Solexa, among others. Array andhybridization sequencing methods are commercially available and/or arebeing developed by Affymetrix, Hyseq, Biotrove, Nimblegen, Illumina, andothers. Methods of sequencing single molecules are being pursued byHelicos based on sequencing by synthesis and U.S. Genomics (amongothers) based on poration.

These methods represent a considerable improvement in throughput overpast methods, in some regards. And they promise considerable improvementin economy as well. However, currently they are expensive to implementand use, the are limited to relatively short reads and, althoughmassively parallel, they have limitations that must be overcome torealize their full potential.

One particular disadvantage of these methods, for example, is thatsamples must be processed serially, reducing throughput and increasingcost. This is a particularly great disadvantage when large numbers ofsamples are being processed, such as may be the case in clinical studiesand environmental sampling, to name just two applications.

The incorporation of indexing sequences by ligation to random shotgunlibraries has been disclosed in U.S. Pat. Nos. 7,264,929, 7,244,559, and7,211,390, but the direct ligation methods therein disclosed distort thedistribution of the components within the samples (as illustrated inFIG. 4 herein) and therefore are inappropriate for enumeratingcomponents within each sample.

Accordingly, there is a need to improve sample throughput, to lower thecosts of sequencing polynucleotides from many samples at a time, and toaccurately enumerate the components of samples analyzed by highthroughput, parallelized and multiplex techniques.

SUMMARY

It is therefore an object of the present invention to provide sequencingmethods with improved sample throughput. The following paragraphsdescribe a few illustrative embodiments of the invention that exemplifysome of its aspects and features. They are not exhaustive inillustrating its many aspects and embodiments, and thus are not in anyway limitative of the invention. Many other aspects, features, andembodiments of the invention are described herein. Many other aspectsand embodiments will be readily apparent to those skilled in the artupon reading the application and giving it due consideration in the fulllight of the prior art and knowledge in the field.

Embodiments provide multiplex methods for the quantitative determinationof polynucleotides in two or more samples, comprising:

-   -   hybridizing a first primer to polynucleotides in a first sample,        said first primer comprising a first tag sequence and a first        probe sequence specific for a first target sequence, wherein        said first target sequence is 3′ to a variable genetic region;    -   elongating primer templates formed thereby to form a first        population of tagged polynucleotides comprising: said first        primer including said first tag sequence; and sequences of said        variable genetic region;    -   hybridizing a second primer to polynucleotides in a second        sample, said second primer comprising a second tag sequence and        a second probe sequence specific for a second target sequence,        wherein said second target sequence is 3′ to the same variable        genetic region as said first target sequence, wherein further        said second probe sequence may be the same as or different from        said first probe sequence;    -   elongating primer templates formed thereby to form a second        population of tagged polynucleotides comprising: said second        primer including said second tag sequence; and sequences of said        variable genetic region;    -   mixing said first and second populations together;    -   determining sequences of polynucleotides comprising tag        sequences and the sequences of the variable genetic element in        said mixture;    -   from the tag sequences comprised in the polynucleotide sequences        thus determined identifying the sample in which polynucleotide        sequences occurred;    -   from the sequences of the variable genetic region comprised in        the polynucleotide sequences thus determined identifying        particular variants of said variable genetic element;    -   from this information determining the number of time one or more        given variants occur in each sample, and    -   from the number for each variant in the polynucleotides thus        determined, quantifying said polynucleotides in said samples;    -   wherein said sequences are determined without Southern blot        transfer and/or without size-separating primer extension        products and/or without electrophoresis.

Embodiments provide multiplex methods for the quantitative determinationof polynucleotides in two or more samples, comprising:

-   -   hybridizing a first primer pair to polynucleotides in a first        sample, the first primer of said first primer pair comprising a        first tag sequence and a first probe sequences specific for a        first target sequence and the second primer of said first primer        pair comprising a second tag sequence and a second probe        sequence specific for a second target sequence, wherein the        first and the second probe sequences flank and hybridize to        opposite strands of a variable genetic region;    -   elongating primer templates formed thereby to from a first        population of tagged polynucleotides, each of said        polynucleotides comprising: (a) the sequence of said first        primer of said first primer pair, a sequence of said variable        genetic region, and a sequence complementary to the sequence of        said second primer of said first primer pair or (b) a sequence        complementary to the sequence of said first primer of said first        primer pair, a sequence of said variable genetic region and the        sequence of said second primer of said first primer pair;    -   hybridizing a second primer pair to polynucleotides in a second        sample, the first primer of said second primer pair comprising a        third tag sequence and said first probe sequences specific for        said first target sequence and the second primer of said second        primer pair comprising a fourth tag sequence and said second        probe sequence specific for said second target sequence;    -   elongating primer templates formed thereby to from a second        population of tagged polynucleotides, each of said        polynucleotides comprising: (a) the sequence of said first        primer of said second primer pair, a sequence of said variable        genetic region, and a sequence complementary to the sequence of        said second primer of said second primer pair or (b) a sequence        complementary to the sequence of said first primer of said        second primer pair, a sequence of said variable genetic region        and the sequence of said second primer of said second primer        pair;    -   mixing said first and second populations together;    -   determining sequences of polynucleotides in said mixture,        comprising the tag sequences and the variable genetic element;    -   from the tag sequences comprised in the polynucleotide sequences        thus determined identifying the sample in which polynucleotide        sequences occurred;    -   from the sequences of the variable genetic region comprised in        the polynucleotide sequences thus determined identifying        particular variants of said variable genetic element;    -   from this information determining the number of times given        variants occur in each sample, and    -   from the number for each variant in the polynucleotides thus        determined, quantifying said polynucleotides in said samples,    -   wherein said sequences are determined without Southern blot        transfer and/or without size-separating primer extension        products and/or without electrophoresis.

Embodiments provide methods in accordance with any of the foregoing orthe following wherein given polynucleotide sequences in a sample isquantified by a method comprising normalizing the number occurrencesdetermined for the given sequence. In embodiments the number ofoccurrences is normalized by dividing the number of occurrencesdetermined for the given polynucleotide sequence by the total number ofoccurrences of polynucleotide sequences in the sample. In embodimentsthe given polynucleotide sequences is that of a given variant of avariable genetic region and, in embodiments, the quantity of the givenvariant in the sample is normalized by dividing the number ofoccurrences of that variant by the total number of occurrences of allvariants of the variable genetic region in the sample.

Embodiments provide a multiplex method for determining polynucleotidesequences in two or more samples, comprising: attaching a first tagsequence to one or more polynucleotides of a first sample; attaching asecond tag sequence different from said first tag sequence to one ormore polynucleotides of a second sample; mixing the taggedpolynucleotides of said first and second samples together; determiningsequences of said polynucleotides comprising said first and said secondtags; and identifying said first and second tags in said sequences;thereby identifying sequences of said polynucleotides of said firstsample and second samples, wherein said sequences are determined withoutSouthern blot transfer and/or without size-separating primer extensionproducts and/or without electrophoresis.

Embodiments provide a multiplex method for determining polynucleotidesequences in two or more samples comprising:

-   -   attaching a first tag sequence, t₁, to P₁₋₁ through P_(1-n1)        polynucleotides in a first sample, thereby to provide a first        plurality of polynucleotides tagged with said first tag, tiPii        through t₁P_(1-n1);    -   attaching a second tag sequence, t₂, to P₂₋₁ through P_(2-n2)        polynucleotides in a second sample, thereby to provide a second        plurality of polynucleotides tagged with said second tag, t₂        P₂₋₁ through t₂P_(2-n2);    -   mixing together said polynucleotides tagged with said first and        said second tags;    -   determining sequences of polynucleotides comprising said tags in        said mixture;    -   identifying said first and second tags in said sequences and;    -   by said first tag identifying polynucleotide sequences of said        first sample and by said second tag identifying polynucleotide        sequences of said second sample;    -   wherein said sequences are determined without Southern blot        transfer and/or without size-separating primer extension        products and/or without electrophoresis.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the number of said polynucleotides in said firstsample, n1, is any of 2, 5, 10, 25, 50, 100, 150, 200, 250, 500, 1,000,1,500, 2,000, 2,500, 5,000, 7,500, 10,000, 12,500, 15,000, 17,500,20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 75,000, 100,000,150,000, 200,000, 250,000, 500,000, 1,000,000 or more, and the number ofsaid polynucleotides in said second sample, n₂, is any of 2, 5, 10, 25,50, 100, 150, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 7,500,10,000, 12,500, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000,75,000, 100,000, 150,000, 200,000, 250,000, 500,000, 1,000,000 or more.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the number of said samples and of said different tagstherefor is 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 500, 1,000,2,500, 5,000, 10,000 or more.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the tags are nucleotide sequences that are 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 nucleotides long orany combination thereof.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the tags are incorporated into said polynucleotidesby a step of ligation, provided that the step of ligation does notresult in biasing.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the tags are incorporated into said polynucleotidesby a step of ligation and/or by a step of amplification.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein said tags are comprised in primers for amplificationand are incorporated into said polynucleotides by amplification usingsaid primers.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein said tags are incorporated into said polynucleotidesby a process comprising a step of cloning into a vector.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the tags are comprised in adapters for amplificationand said adapters are ligated to polynucleotides in said samples.Embodiments provide a method in this regard, wherein further, saidpolynucleotides ligated thereby to said tags are amplified via saidadapters. Embodiments provide a method in this regard, wherein further,said adapters comprise a moiety for immobilization. In embodiments saidmoiety is a ligand; in embodiments it is biotin. Embodiments provide amethod in this regard, wherein further, said tags are comprised onadapters for bead emulsion amplification. In embodiments the adaptersare suitable for use in a sequencing system of 454 Life Sciences orother sequencing system in which bead emulsion amplification is carriedout.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the primer for amplification comprises a sequence forPCR amplification, linear amplification, transcriptional amplification,rolling circle replication, or QB replication.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the primer for amplification comprises a sequence forPCR amplification.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein each of said polynucleotides is disposed individuallyon a bead isolated from other polynucleotides.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein each of said polynucleotides is disposed individuallyon a bead isolated from other said polynucleotides, is amplified whiledisposed therein, and the amplification products thereof also aredisposed on said bead.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein each of said polynucleotides is disposed individuallyon a bead isolated from other said polynucleotides, is amplified whiledisposed therein, the amplification products thereof also are disposedon said bead, and each said bead is disposed individually in a wellisolated from other said beads.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the sequences are determined by pyrosequencing.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein said samples are biological samples, each comprisingone or more species.

Embodiments provide a method according to any of the foregoing or thefollowing, at least one sequence of said polynucleotides is specific toa particular organism.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein said sequences comprise a variable 16S rRNA sequence.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein said sequences comprise a variable 18S rRNA sequence,a variable rRNA ITS sequence, a mitochondrial sequence, a microsatellitesequence, a metabolic enzyme sequence, and/or a genetic diseasesequence.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the samples are microbial community samples.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the samples are microbial community samples forclinical analysis of a patient.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein samples are microbial community environmentalsamples.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the samples are microbial community soil samples.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the samples are microbial community water samples.

Embodiments provide a method according to any of the foregoing or thefollowing, wherein the samples are samples for SNP analysis.

Embodiments provide a method according is any of the foregoing or thefollowing, wherein the samples are samples for genotyping.

Embodiments provide a multiplex method according to any of the foregoingor the following for determining polynucleotide sequences of two or moresamples, comprising,

-   -   amplifying polynucleotides of a first sample to produce first        amplified polynucleotides comprising a first tag sequence;    -   separately amplifying polynucleotides of a second sample to        produce second amplified polynucleotides comprising a second tag        sequence different from said first tag sequence;    -   wherein the amplification products arising from different        individual polynucleotides are spatially separated from one        another;    -   mixing together amplicons of said first and second samples;    -   distributing the amplicons in the mixture into spatially        distinct locations; sequencing the amplicons thus distributed        using one or more primers that hybridize 5′ to said tag        sequences;    -   identifying said tag sequences in the sequences of        polynucleotides thus determined; and    -   identifying by said tags polynucleotides of said first sample        and polynucleotides of said second sample.

Embodiments provide a method according to any of the foregoing or thefollowing, comprising,

-   -   (a) for each sample separately: isolating polynucleotides to be        sequenced, ligating said polynucleotides to a common adaptor        comprising a tag sequence, and capturing individual ligated        polynucleotides onto individual beads under conditions that        provide predominately for the immobilization of 0 or 1 molecule        per bead;    -   (b) thereafter mixing together said beads comprising said        polynucleotides.

Embodiments provide a method according to any of the foregoing or thefollowing, further comprising, amplifying bead-immobilizedpolynucleotides in droplets of an emulsion thereby to clonally amplifysaid individual polynucleotides on said beads, wherein amplificationcomprises amplification of said tag sequence.

Embodiments provide a method according to any of the foregoing or thefollowing, further comprising, distributing individual dropletscontaining said amplified polynucleotides into wells under conditionsthat provide predominantly for 0 or 1 droplet per well, determining inindividual wells the sequences of polynucleotides comprising said tagsequences, and by said tag sequences identifying polynucleotides of saidfirst and said second samples.

In embodiments the invention provides methods in accordance with any ofthe foregoing or the following, for any one or more of detecting,monitoring, profiling, prognosticating, and/or diagnosing a disorder,disease, or the like.

In embodiments the invention provides methods in accordance with any ofthe foregoing or the following, for analyzing the composition,diversity, stability, dynamics, and/or changes in agricultural, food,biosecurity, veterinary, clinical, ecological, zoological,oceanological, and/or any other sample comprising one or morepolynucleotides.

Embodiments provide kits comprising a plurality of two or more primers,each primer in said plurality comprising a tag sequence and a probesequence specific to a target sequence, wherein:

-   -   (A) in each of said primers the probe sequence is 3′ to the tag        sequence, but not necessarily adjacent thereto;    -   (B) in each of said primers: the tag sequence is different from        the tag sequence of the other in the plurality; the tag sequence        is not the complementary sequence to any other tag sequence in        the plurality; the tag sequence does not contain any        homodinucleotide sequences; the junction sequences between the        tag sequence and the adjacent parts of the primer, if any, is        not a homodinucleotide sequence;    -   (C) in each of said primers the probe sequence is complementary        to the target sequence and the target sequence is located 3′ to        a variable genetic region, and    -   (D) each of said primers is disposed separately from the others        in containers in said kit.

Embodiments provide kits in accordance with any of the foregoing or thefollowing, wherein each of said primers further comprises a primingsequence 5′ to the tag sequence but not necessarily adjacent thereto,and the priming sequence is the same in all of said primers, said kitfurther comprising a primer complimentary to and effective forpolymerization from said priming sequence.

Embodiments provides kits comprising a plurality of two or more primerspairs, each primer in said plurality comprising a tag sequence and aprobe sequence specific to a target sequence, wherein:

-   -   (A) in each of said primer the probe sequence is 3′ to the tag        sequence, but not necessarily adjacent thereto;    -   (B) in each of said primers: the tag sequence is different from        the tag sequence of the other in the plurality; the tag sequence        is not the complementary sequence to any other tag sequence in        the plurality; the tag sequence does not contain any        homodinucleotide sequences; the junction sequences between the        tag sequence and the adjacent parts of the primer, if any, is        not a homodinucleotide sequence;    -   (C) in each of said, primers the probe sequence is complementary        to the target sequence,    -   (D) in each primer pair the probe sequences are specific to        target sequences that flank a variable genetic region;    -   (E) each of said primers is disposed separately from the others        in said kit.

Embodiments provides kits in accordance with any of the foregoing or thefollowing, wherein, the primers further comprise a priming sequence 5′to the tag sequence but not necessarily adjacent thereto, the primingsequence either is the same in all the primers, or one member of eachpair has the same first priming sequence and the second member of eachpair has the same second priming sequence, said kit further comprisingdisposed separately from one another in one or more containers one ormore primers complementary to and effective for elongation from saidpriming.

Embodiments provide a kit useful in methods according to any of theforegoing or the following, comprising a set of primers and/or adapters,wherein each primer and/or adapter in said set comprises a tag sequenceand a primer sequence. In embodiments the primers and/or adaptersfurther comprise a moiety for immobilization. In embodiments the primersand/or adapters comprise biotin. In embodiments the primers and/oradapters in the set comprise all tag sequences defined by 2, 3, 4, 5, 6,7, or 8 base polynucleotide sequences, wherein each of said primersand/or adapters are disposed in containers separate from one another. Inembodiments there are 1-5, 3-10, 5-15, 10-25, 20-50, 25-75, 50-100,50-150, 100-200, 150-500, 250-750, 100-1000, or more different tagsequences disposed separately from one another, so as to be useful foruniquely tagging said number of different samples. In embodiments theprimers and/or adapters are suitable for use as 454 Life Sciencesamplification adapters and/or primers. In embodiments the primers and/oradapters further comprise any one or more of a primer sequence for anyone or more of a 16S rRNA sequence, an 18S rRNA sequence, an ITSsequence, a mitochondrial sequence, a microsatellite sequence, ametabolic enzyme sequence, a genetic disease sequence, and/or any othersequence for amplification or analysis.

In embodiments the invention provides a kit, in accordance with any ofthe foregoing or the following, comprising a set of primers and/oradapters for use in a method according to any of the foregoing or thefollowing, wherein each primer and/or adapter in said set comprises atag sequence, the tag sequence of each of said primers and/or adaptersis different from that of the other primers and/or adapters in said set,the primers and/or adapters further comprise a priming sequence that isthe same in all of the primers and/or adapters in said set, the tagsequences are located 5′ to the priming sequence and the differentprinters and/or adapters comprising each different tag sequence aredisposed separately from one another. In embodiments the tags are anynumber of bases long. In embodiments the tags are 2, 3, 4, 5, 6, 8, 10,12 bases long. In embodiments the tags are 4 bases long. In embodimentsthe priming sequence is specific to any target polynucleotide ofinterest. In embodiments the priming sequence is specific to a sequencein 16S rRNA. In embodiments the tags differ from each other by at least2 bases. In embodiments the tags do not contain polynucleotide tractswithin the tag. In embodiments the tags do not containhomo-polynucleotide tracts within or at the junction of the tag and PCRprimer. In embodiments the tags do not contain polynucleotide tractswithin or at the junction of the tag and emulsion PCR adapter. Inembodiments, the tags are not reverse compliments of each other.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram showing a general embodiment of theinvention. A plurality of samples (S₁, S₂ through S_(j)) is showntopmost in the Figure. Each sample is comprised of a plurality ofpolynucleotides (P₁₋₁ to P_(1-n1) in S₁; P₂₋₄ to P_(2-n2) in S₂; throughP_(j-1) to P_(j-nj)). The polynucleotides in each sample are labeledseparately with a tag polynucleotide sequence, all the polynucleotidesin a given sample being tagged (in this illustration) with a single tagsequence, designated in the figure as T₁ for S₁, T₂ for S₂, throughT_(j) for S_(j). The individual tagged polynucleotides are denotedaccordingly. The tagged polynucleotides in each sample are designatedcollectively, for each sample, T₁S₁, T₂S₂, through T_(j)S_(j). Thetagged polynucleotides from the samples are mixed together to form amixture, designated M. The mixture is sequenced typically by a massivelyparallel sequencing method. The tag sequences are identified in the datathus obtained. The sequences are grouped by tag. The sequences from theindividual samples are thereby identified.

FIG. 2A is a diagram depicting step I in the multitag sequencing ofmicrobial community samples using a tagged 16S forward and reverseprimer-linker pairs for PCR amplification, (a) represents the Forward16S rRNA primer with Tag l and Emulsion PCR Linker, (b) represents the16S rRNA sequence, (c) represents the Reverse 16S rRNA primer with Tag jand Emulsion PCR Linker, (d) represents the Amplified 16S rRNA sequencewith Forward and Reverse Tags ij, (e) represents the Emulsion PCR Bead,(f) represents the pyrosequencing read, (g) represents the well inpicoliter plate, (h) represents a Unique tag, (i) represents AmplifiedCommunity 1, (j) represents Amplified Community 2, and (k) representsAmplified Community n. Step 1 involves the amplification of themicrobial community from each sample using uniquely tagged universalprimers-linkers. In step 1, different samples are amplified separately,using 16S rRNA specific adapter-tag-primers with a different tag foreach sample.

FIG. 2B is a diagram depicting the Emulsion PCR reaction beads randomlyarrayed into picoliter plate. In step 2 in the process, the PCR productsfrom all the samples are mixed, immobilized on beads, distributed intowells of the picoliter plate, and emulsion PCR amplified.

FIG. 2C is a diagram depicting the pyrosequencing process from eachoutside adapter in each well of the picoliter plate. Each reaction readssequence from the adapter, through the unique tags and the associatedsequence of the tagged sample.

FIG. 2D is a diagram depicting the algorithmic sorting of thePyrosequencing reads using the individual tag sequence and a portion ofthe printer sequence. (1) represents the sequence reads from sample 1,(m) represents the sequence reads from sample 2, and (n) represents thesequence reads from sample n.

FIG. 2E is a diagram depicting the identification of microbial taxa bycomparing the sequence reads for each sample against the 16S rRNAsequence database and then normalize abundance in each taxa with respectto the total reads in that particular sample. (o) represents thenormalized species histogram derived the pyrosequencing reads obtainedfrom sample 1, (p) represents the normalized species histogram derivedthe pyrosequencing reads obtained from sample 2, (q) represents thenormalized species histogram derived the pyrosequencing reads obtainedfrom sample n.

FIG. 3 is the species distribution in (A) Controls, (B) Crohns, and (C)Ulcerative colitis samples determined by the 454 Life Sciencepyrosequencing process. Each bar in the histogram is the averagenormalized abundance of that taxa in each disease state. Each sample wasrun in a separate well on the picoliter plate using the 454 16 wellmask.

FIGS. 4A and 4B show an example of the distortion of the components of acomplex mixture caused by ligating the Emulsion PCR adapters onto PCRamplicons. FIG. 4A shows the size distribution of PCR amplicons insample 309 before ligation and FIG. 4B shows the size distribution ofsample 309 after ligation.

FIG. 5 is an example of the normalized taxa abundances in duplicatesamples determined by Multitag pyrosequencing after direct ligation ofthe emulsion PCR adapters.

FIG. 6 shows all possible hexameric polynucleotide tags within whichthere are no dinucleotide repeats and no tag is the reverse complementof any other tag.

FIGS. 7A-D show 96 tagged adaptor primers in which there are nodinucleotide repeats in the tags, no dinucleotide repeats at thejunction of the tags and the tags are not reverse complements of oneanother. In each case 5 bases of the primer also can be used to identitysamples. 7A and 7B show the forward primers. 7C and 7D show the reverseprimers.

GLOSSARY

The meanings ascribed to various terms and phrases as used herein areillustratively explained below.

“A” or “an” means one or more; at least one.

“About” as used herein means roughly, approximately. Should a precisenumerical definition be required, “about” means +/−25%.

“Adapter” means a polynucleotide sequence used to either attach singlepolynucleotide fragments to beads and/or to prime the emulsion PCRreaction and/or as a template to prime pyrosequencing reactions.

“ALH” is used herein to mean amplicon length heterogeneity.

“Amplicon” is used herein to refer to the products of an amplificationreaction.

“Clonally amplified” is used herein generally to mean amplification of asingle starting molecule. Typically it also refers to the clusteringtogether of the amplification products, isolated from otheramplification templates or products.

“dsDNA” means double stranded DNA.

Dysbiosis means a shift in a the species and abundance of species in amicrobial community.

“Flanking” generally is used to mean on each side, such as on the 5′ andthe 3′ side of a region of a polynucleotide—with reference to the 5′ andthe 3′ ends of one or the other stand of a double strandedpolynucleotide. Forward and reverse primers for amplifying a region of apolynucleotide by PCR, for instance, flank the region to be amplified.

“Microbial community sample” is used herein to refer to a sample,generally of a biological nature, containing two or more differentmicrobes. Microbial community samples include, for instance,environmental samples, as well as biological samples, such as samplesfor clinical analysis. The term applies as well to preparations, such asDNA preparations, derived from such samples.

“Multiplex sequencing” herein refers to sequencing two or more types orsamples of polynucleotides in a single reaction or in a single reactionvessel.

“PCO” means principal coordinates analysis.

“PCA” means principal component analysis.

“Picotiter plate” means a plate having a large number of wells that holda relatively small volume, typically more wells than a 96-wellmicrotiter plate, and smaller volumes than those of a typical 96-wellmicrotiter plate well.

“Primer” means a polynucleotide sequence that is used to amplify PCRproducts and/or to prime sequencing reactions.

“ssDNA” means single stranded DNA.

“Tag,” “Tag sequence,” etc. means typically a heterologous sequence,such as a polynucleotide sequence that identifies another sequence withwhich it is associated as being of a given type or belonging to a givengroup.

“Variable genetic region” as used herein means a genetic region thatvaries, such as between individuals of a species and between species.The phrase does not denote a specific length, but, rather is used todenote a region comprising a variation the exact length of which mayvary and may differ in different contexts. As to a double strandedpolynucleotide, the term includes one or the other and both stands ofthe region, and may be used to refer to one, the other, or to bothstrands, and it will generally be clear from the context which is meant.A specific example of a genetic region that varies between individuals,provided for illustration only, is a genetic region that contains an SNP(single nucleotide polymorphism) site. By variable genetic region inthis regard is meant a region containing the SNP site. Differentsequences of the SNP in this regard constitute the variants of thevariable genetic region. A specific example of a variable genetic regionthat differs between species is the genes for 16S RNA which varycharacteristically between microbes and can be used to identify microbesin mixed community samples as described in greater detail in some of theexamples herein.

DESCRIPTION OF THE INVENTION

In certain aspects and embodiments the invention relates to multiplexsequencing analysis using tags. In various aspects and embodiments ofthe invention in this regard the invention provides methods forsequencing two or more samples simultaneously in a mixture with oneanother, wherein each sample is first linked to a sample-specificsequence tag, the tagged samples are mixed and sequenced, and thesequences from each sample then are identified by their respectivesample-specific sequence tags.

FIG. 1 provides a general depiction of various aspects and embodimentsof the invention in this regard, and the figure is discussed by way ofillustration below with reference to sequencing DNA from differentsamples. A plurality of samples (S₁, S₂, through S_(j)) is shown topmostin the Figure. Each sample is comprised of a plurality ofpolynucleotides (P₁₋₁ to P_(1-n1) in S₁; P₂₋₁ to P_(2-n2) in S₂; throughP_(j-1) to P_(j-nj)). The polynucleotides in each sample are labeledseparately with a tag polynucleotide sequence, all the polynucleotidesin a given sample being tagged (in this illustration) with a single tagsequence, designated in the figure as T₁ for S₁, T₂ for S₂, throughT_(j) for S_(j). The individual tagged polynucleotides are denotedaccordingly. The tagged polynucleotides in each sample are designatedcollectively, for each sample, T₁S₁, T₂S₂ through T_(j)S_(j). The taggedpolynucleotides from the samples are mixed together to form a mixture,designated M_(i). The mixture is sequenced typically by a parallelsequencing method. The tag sequences are identified in the data thusobtained. The sequences are grouped by tag. The sequences from theindividual samples are thereby identified.

In embodiments tags are 3 to 30, 4 to 25, 4 to 20 base long sequences.In embodiments the tags are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36 nucleotides long or any combination thereof.

In embodiments there are 1-6, 6-12, 10-15, 10-20, 15-25, 20-40, 25-50,25-75, 50-100, 50-150, 100-200, 100-250, 50-250, 100-500, 500-1,000,100-1,000, 500-5,000, 100-10,000, 1,000-25,000, 500-50,000, 100-100,000,1-1,000,000 or more samples, tagged, respectively, with 1-6, 6-12,10-15, 10-20, 15-25, 20-40, 25-50, 25-75, 50-100, 50-150, 100-200,100-250, 50-250, 100-500, 500-1,000, 100-1,000, 500-5,000, 100-10,000,1,000-25,000, 500-50,000, 100-100,000, 1 1,000,000 or more differenttags.

In embodiments the sequences are determined without the use of gelelectrophoresis. In embodiments the sequences are determined without theuse of transfer of sequences from a gel onto a membrane or a filter forhybridization. In embodiments, sequences are determined by a parallelsequencing method. In embodiments the sequences are determined bypyrosequencing, sequencing by synthesis, hybridization sequencing,subtractive sequencing, pore sequencing or direct read sequencing.

In embodiments the tags are incorporated into polynucleotides in samplesfor sequencing by a step of ligation and/or by a step of amplification.

In embodiments the tags are comprised in primers for amplification.

In embodiments the tags are comprised in primers for PCR amplification,transcription amplification, rolling circle amplification, oramplification by Qβ replicase.

In embodiments the tags are comprised in emulsion PCR adapters andprimers for amplification.

In embodiments the tags are incorporated by a step of cloning into avector.

In embodiments the samples are microbial community samples. Inembodiments the samples are clinical samples. In embodiments the samplesare environmental samples. In embodiments the samples are samples forSNP analysis. In embodiments the samples are samples for genotyping. Inembodiments the sequences are determined in one or more picotiterplates.

In embodiments the samples are fragmented genomic DNAs. In embodimentsthe samples are fragmented Bacterial genomic DNA, Archae genomic DNA,Fungal genomic DNA, Eukaryotic genomic DNA, chloroplast DNA, and/ormitochondrial DNA. In embodiments the samples are cDNAs. In embodimentsthe samples are Eukaryotic cDNA, Bacterial cDNA, Archae cDNA, and/orFungal cDNA. In embodiments the tags are incorporated by a step ofligation and/or by a step of amplification.

In embodiments the samples are for any one or more of detecting,monitoring, profiling, prognosticating, and/or diagnosing a disorder,disease, or the like.

In embodiments the samples are for analyzing the composition, diversity,stability, dynamics, and/or changes in agricultural, food, biosecurity,veterinary, clinical, ecological, zoological, oceanological, and/or anyother sample comprising one or more polynucleotides.

In embodiments the sequences are determined in wells of a titer plate.In embodiments the sequences are determined in one or more picotiterplates having a mask. In embodiments the sequences are determined in onemore picotiter plates having a mask, wherein the mask defines 2, 4, 8,16, 32, 64 or more compartments.

By way of illustration to a 454 picotiter plate, in embodiments thereare about 120,000 templates/plate and the read length averages about 250bases per template. In embodiments relating thereto there are 10 tags of4 bases per 1/16 plate, 160 tags total, an average of about 750templates per tag (and per sample), and about 187,500 bases sequencedper tag (and per sample).

In embodiments there are about 260,000 templates/plate and the readlength averages about 250 bases per template. In embodiments relatingthereto, there are 12 tags of 4 bases per 1/8 plate, 96 samples total,an average of about 2,708 templates per tag (and per sample) and about677,083 bases of sequence per tag (and per sample).

In embodiments there are about 400,000 templates/plate and the readlength averages about 250 bases per template. In embodiments relatingthereto, there are 96 tags of 6 bases for 96 samples per plate, about4,166 templates per tag (and per sample) and about 1,041,666 bases ofsequence per tag (and per sample).

In embodiments the tags are 10 base long sequences, there are 192different tags, and the samples are analyzed in microtiter plate format.

In embodiments the invention provides algorithms for deconvolving, froma mixture of sequences from two or more samples, the sequences of thesamples in the mixture by identifying sample-specific tags in thesequences, grouping the sequences by the tags thus identified, therebygrouping together the sequence from each of said samples, apart from oneanother.

In embodiments the invention provides algorithms for deconvolving, froma mixture of sequences from two or more samples, the sequences of thesamples in the mixture by identifying sample-specific tags in sequences,as follows:

-   -   1. Road all sequence reads into an array;    -   2. Search the beginning of each sequence read and identify the        tag;    -   3. Build an associative array linking tag with sequence read;    -   4. Sort the keys for the associate array;    -   5. Associate each key with the corresponding sample;    -   6. Pool all sequence reads for each sample;    -   7. Analyze each sample separately.    -   8. Normalize the abundance of each component within each samples        with respect to the total reads within that sample.

In embodiments the algorithm can be implemented in any programminglanguage. In embodiments the algorithm is implemented in C, C++, JAVA,Fortran, or Basic. In embodiments the algorithm is implemented as a PERLscript.

In embodiments the invention provides kits for multiplex sequencing asdescribed herein, comprising a set of primers and/or adapters, whereineach primer and/or adapter in said set comprises a tag sequence, aprimer sequence and/or an emulsion PCR adapter. In embodiments theprimers and/or adapters further comprise a moiety for immobilization. Inembodiments the primers and/or adapters comprise biotin. In embodimentsthe primers and/or adapters in the set comprise all tag sequencesdefined by 2, 3, 4, 5, 6, 7, or 8 base polynucleotide sequences, whereinsaid primers and/or adapters comprising different tag sequences aredisposed in containers separate from one another. In embodiments thereare 1-5, 3-10, 5-15, 10-25, 20-50, 25-75, 50-100, 50-150, 100-200,150-500, 250-750, 100-1000, or more different tag sequences disposed,separately from one another, so as to be useful for uniquely taggingsaid number of different samples. In embodiments the primers and/oradapters are suitable for use as 454 Life Sciences amplificationadapters and/or primers. In embodiments the primers and/or adaptersfurther comprise any one or more of a primer sequence for any one ormore of a 16S rRNA sequence, an 18S rRNA sequence, an ITS sequence, amitochondrial sequence, a microsatellite sequence, a metabolic enzymesequence, a genetic disease sequence, and/or any other sequence foramplification or analysis.

EXAMPLES

The present invention is additionally described by way of the followingillustrative, non-limiting examples.

Example 1 Sequencing Using the 454 Pyrosequencing System

454 Life Sciences, a subsidiary of Roche Diagnostics, provides a devicefor pyrosequencing approximately 100,000,000 bases of about 400,000different templates in a single run on a single picotiter plate. Thecompany also provides masks that allows for the processing 2, 4, 8, or16 different samples on one plate. At maximum capacity using the maskedplate, the system provides about 1 million bases of sequence data onabout 4,000 templates for each of 16 samples.

The general process of sequencing using the 454 system is generally asfollows: isolate DNA; optionally fragment the DNA; optionally render theDNA double stranded; ligate the DNA to adaptors; separate the strands ofthe dsDNA, bind the ssDNA to beads under conditions that result in apreponderance of beads that have either no DNA molecule bound to them ora single molecule of DNA bound to them; capture the beads in individualdroplets of an emulsion of a PCR reaction mix in oil; carry out a PCRreaction on the emulsion-encapsulated bead-DNAs (whereby amplificationproducts are captured on the beads); distribute the amplificationproducts into picoliter wells so that there is either no bead in a wellor one bead; and carry out pyrosequencing on all the beads in all thewells in parallel.

Example 2 Multiplex Pyrosequencing Using 96 Tagged Adapter-PCR Primers

454 Life Sciences, a subsidiary of Roche Diagnostics, provides a devicefor pyrosequencing approximately 100,000,000 bases of sequence for about400,000 different templates in a single run on a single picotiter plate.At maximum capacity using the plate, the system provides about 10million bases of sequence data for each of about 4,000 templates foreach of 96 multitagged samples. In this example the 96 tags are 6 basesin length and are used along with 6 bases of the forward or reverseprimer to identify the reads that belong with each of the 96 individualsamples (see FIG. 2).

Example 3 Multitag Pyrosequence Analysis of Microbial Community Samples

Various aspects and embodiments of the invention herein described areillustrated by way of the following general example relating to“ecogenomic” analysis of microbial diversity in biological samples.

The ability to quantify the number and kinds of microorganisms within acommunity is fundamental to the understanding of the structure andfunction of an ecosystem, as discussed in, for instance, Pace 1997 andTheron and Cloete 2000. Traditionally, the analysis of microbialcommunities has been conducted using microbiological techniques, butthese techniques are limited. For instance they are not useful for themany organisms that cannot be cultivated (Ritchie, Schutter et al. 2000;Spring, Schulze et al. 2000). Even for those organisms that can becultured, these techniques provide little information with which toidentify individual microbes or characterize their physiological traits.(Morris, Bardin et. al. 2002).

Recent advances in molecular techniques have overcome some of thesedisadvantages, and have enabled the identification of many more taxa inmicrobial communities than traditional microbial techniques. Theseadvances have provided considerable insight into the expression of keyfunctions in species in microbial communities. (Pace 1997; Suzuki 1998;Amann 2000; Frischer, Danforth et al. 2000; Ritchie, Schutter et al.2000; Spring, Schulze et al. 2000). Among these molecular techniques areDenaturing Gradient Electrophoresis (DGGE), Temperature Gradient GelElectrophoresis (TGGE), Temporal Temperature Gradient GelElectrophoresis (TTGE), Terminal-Restriction Fragment LengthPolymorphism Single Strand Conformation Polymorphism (SSCP), and LengthHeterogeneity PCR (LH-PCR) (Frischer, Danforth et al. 2000; Theron andCloete 2000; Mills, Fitzgerald et al. 2003; Seviour, Mino et al. 2003;Klaper and Thomas 2004).

Among these, LH-PCR is probably the best technique for fingerprinting.It is inexpensive, fast, and can be used routinely to screen severalhundred samples a day. It is useful as a routine survey tool that can beused to monitor the dynamics of natural soil microbial communities, andto quickly identify samples of interest by PCO analysis. LH-PCR has beenused to extensively assess natural variation in bacterial communities byprofiling the amplified variable regions of 16S rRNA genes in mixedmicrobial population samples, using PAGE. (See Mills 2000; Litchfieldand Gillevet 2002; Lydell, Dowell et al. 2004). The LH-PCR products ofthe individual species in the population give rise to distinct bands inthe gels. The “peak area” of each band is proportional to the abundanceof the species in the community. LH-PCR of 16S rRNA variable regions hasbeen used quite successfully to estimate species diversity inbacterioplankton communities, in particular. (See Suzuki, Rappe et al,1998; Ritchie, Schutter et al. 2000).

Community functionality cannot be determined directly from 16S rRNAclone data, however, it must be inferred from the data by phylogeneticanalysis. Furthermore, LH-PCR and other fingerprinting technologies,while powerful tools for monitoring population dynamics, cannot identifyindividual species in a community. For this, fingerprintinginvestigations must be followed up by library construction, cloning,sequencing, and phylogenetic analysis. (Fitzgerald 1999; McCraig 1999;Spring, Schulze et al. 2000; Theron and Cloete 2000; Litchfield andGillevet 2002; Bowman and McCuaig 2003; Kang and Mills 2004; Eckburg,Bik et al. 2005). Identifying species of a fingerprinting study, thus,is a considerable undertaking that is inconvenient, time-consuming,expensive and subject to technical limitations.

Grouping samples can, to some extent, reduce the cost, time, and expenseof such analyses. For instance, PCO analysis of LH-PCR data can be usedto group samples with similar profiles for batch cloning and sequencing.Combining the samples this way reduces the time, expense, and workinvolved in analyzing the samples. Sequencing of at least 300 randomclones is required to identify the bacterial components of the pooledsample down to 1% of the total bacterial populations in typical samples.This level of resolution is similar to that of ALH fingerprinting.Originally a novel approach, pooling similar samples prior to cloningand sequencing has proven to be robust and effective.

In classic community studies in the literature (Eckburg, Bik et al.2005), environmental samples are assayed independently. Then the clonesequence data from specific classes/groups are statistically analyzedusually using some sort of averaging metric. Analyses of this type canbe extremely costly, especially if the clone libraries are exhaustivelyanalyzed, something that typically involves sequencing thousands ofclones. Moreover, for the “averaging” process to be valid, a requiredfor comparing the mixed populations, the samples must be pooled in equalproportions. While simple in principle, in reality, it is difficult toaccomplish and, even if accomplished, impossible to verify. A newtechnique, based on pyrosequencing, offers advantages that overcome avariety of these drawbacks of the fingerprinting technologies mentionedabove. The method is implemented on an instrument sold by 454 LifeSciences, Inc., a subsidiary of Curagen Sciences, Inc., using reagentsprovided by the same company. In addition, 454 Life Sciences provides acustom service for pyrosequencing.

In this technology, individual DNA molecules are amplified on beads byPCR in individual droplets in an oil-in-water emulsion. Beads then aredeposited individually in wells of picotiter plate. The sequences of theDNAs in the wells are determined in parallel by pyrosequencing. (SeeVenter, Levy et al. 2003 Margulies, Egholm et al. 2005; Poinar, Schwarzet al. 2006). In a typical run, there are about 200,000 templates perplate, an average read length of about 100 bases from each template, anda single-plate run generates about 20 million bases of sequence in asingle four hour run.

Although the technology greatly increases throughput over previousmethods, it is expensive. In particular, the cost per plate is too highfor it to be economically practical to carry out many analyses. Todecrease cost, masks can be used that divide a plate into 16 independentsample zones, so that one plate can be used to process 16 differentsamples, either at the same time or independently. Each 1/16 zoneprovides about 1,000,000 bases of sequence data from about 10,000different templates. While this reduces the cost per sample, theexpenses associated with using this technology remain undesirably high.

Various aspects and embodiments of the present invention can be used tofurther reduce the cost per sample of this technology (as well as othertechniques, as described elsewhere herein). The use of multitaggingtechniques (referred to as, among other things, “Multitag process”) tothe genomic analysis of bacterial populations in according with certainaspects and embodiments of the invention, notably high coveragesequencing of bacterial communities, is referred to herein as “MultitagEcogenomics” and also as “Multitag Ecogenomic Analysis.”

(Several publications use the term “Multiplex Pyrosequencing” (Pourmand,Elahi et al. 2002) to refer to generating a composite signal frommultiple targets that is read as a signature for a specific sample. Theterm is not used to refer to tag-based multiplexing in which sequencesfrom different samples in a mixture are determined and then deconvolvedfrom the mixed sequencing data using sample-specific tags incorporatedduring amplification reactions.)

As described below the Multitag Process in a relatively simple series ofsteps accomplishes everything that otherwise would require not onlycommunity fingerprinting analysis, but also all of the cloning andsequencing processes previously required for high coverage EcogenomicAnalysis using conventional techniques.

By way of illustration, the following example describes the use ofMultitag Ecogenomic Analysis of variable regions of common genes usingtagged universal primers for high coverage analysis of several microbialcommunity samples all at the same time. The analysis is carried out muchas described in general above, and further elaborated on in detailbelow.

Briefly, short tags are added to the 5′ ends of the forward and reversePCR primers normally used for community analysis. These tags can beplaced between the Emulsion PCR adapters and the PCT primers (see FIG.2). A different tag is attached to the primers for each of the samplesto be combined. For instance primers that span a variable region of 16SrRNA genes may be used for analysis of bacterial and archaelcommunities. 16S rRNA-specific primers with 4 base tags are set out inthe Table 1 below. Likewise primers that span a variable region of anITS gene may be used for analysis of fungal communities. It will beappreciated that the choice of these specific primers is not exclusive,and that a wide variety of other primers suitable to other regions foramplification may be employed in much the same manner as descried hereinfor the 16S and ITS genes. Thus, any gene of interest can be used thatprovides conserved primer sites across a community, and sufficientvariation in the region between the primers for the desired resolutionof individual species. Thus, for example, genes specific to functionalpathways such as anaerobic methane oxidation, or sulphur reduction canserve as targets for the amplification reaction, as well as 16S rRNAsequences.

TABLE 1 Name Tag Forward Shared Sequence AGCTAGAG TTT GATCMTGGCTCAGL27FA AGCT AGCTAGAGTTTGATCMTGGCTCAG L27FB AGTC AGTCAGAGTTTGATCMTGGCTCAGL27FC GATC GATCAGAGTTTGATCMTGGCTCAG L27FD GACT GACTAGAGTTTGATCMTGGCTCAGL27FE CTGC CTGCAGAGTTTGATCMTGGCTCAG L27FF CTAG CTAGAGAGTTTGATCMTGGCTCAGL27FG ATGC ATGCAGAGTTTGATCMTGGCTCAG L27FH ATAG ATAGAGAGTTTGATCMTGGCTCAGL27FM ATCT ATCTAGAGTTTGATCMTGGCTCAG L27FO ATAT ATATAGAGTTTGATCMTGGCTCAGReverse Shared Sequence AGCTGCTGCCTCCCGTAGGAGT 355RA AGCTAGCTGCTGCCTCCCGTAGGAGT 355RB AGTC AGTCGCTGCCTCCCGTAGGAGT 355RC GATCGATCGCTGCCTCCCGTAGGAGT 355RD GACT GACTGCTGCCTCCCGTAGGAGT 355RE CTGCCTGCGCTGCCTCCCGTAGGAGT 355RF CTAT CTATGCTGCCTCCCGTAGGAGT 355RG ATGCATGCGCTGCCTCCCGTAGGAGT 355RH ATAT ATATGCTGCCTCCCGTAGGAGT 355RM ATCTATCTGCTGCCTCCCGTAGGAGT 355RO ATAC ATACGCTGCCTCCCGTAGGAGT

Table 1 shows a 16S rRNA-specific primer with a variety of 4 base tagsequences attached. As described herein such primers are useful foramplifying 16S rRNAs in several samples that can then be sequencedtogether. The 16S rRNA in each sample is amplified using a differenttag, but the same 16S primer sequence. The amplified rRNA sequences fromthe samples are combined and sequenced together. The rRNA sequences fromthe different samples then are identified and sorted out by their 4 basetag sequence plus the first 4 bases of each primer. It is to beappreciated that the sequences downstream of the shared 16S primersequence will differ among the samples, as well as the tag sequence.

In each case, the samples are individually amplified. The resultingamplicons comprise the primer sequences including the tags. Since uniquetags are used for each sample, the tags in the amplicons from eachsample will be different. The amplified DNAs are then pooled andsequenced by pyrosequencing as described above. The sequence data from arun is analyzed, in part, by grouping together all the sequences havingthe same tag. In this way, the sequences from each sample aredemultiplexed from the sequencing data obtained from the mixture.

The working of the invention in this regard is illustrated by thefollowing simulation, carried out using conventionally obtainedpopulation data front cold seep samples. The algorithm for sequenceanalysis uses a PERL script to extract the first 100 bases of sequence.It then analyzes all the 100 bases sequences using a custom RDP PERLscript. The script works as follows:

-   -   1. Read all sequence reads into an associate array (Hash 1);    -   2. Extract 100 base subsequences from the beginning of each        sequence read;    -   3. Create an associate array (Hash 2) of the sequences;    -   4. Perform a Blast search of the RDP database with Hash 1;    -   5. Perform a Blast search of the RDP database with Hash 2;    -   6. Compare the identifications for the original sequence        (Hash 1) and the subsequence (Hash 2);    -   7. Compile a list of similar identifications for Hash 1 and Hash        2;    -   8. Compile a list of different identifications tor Hash 1 and        Hash 2;    -   9. Calculate the percentage of similar identifications.

As shown below, there is virtually no difference at the class level inthe microbial diversity generated by the sequencing simulation and thatderived directly from the 16S rRNA sequences in the data base:

TABLE 2 First 16S RDP Class 100mer rRNA ALPHA_SUBDIVISION 3.6% 3.6%ANAEROBIC_HALOPHILES 3.6% 3.6% BACILLUS-LACTOBACILLUS-STREPTOCOCCUS 3.6%3.6% SUBDIVISION BACTEROIDES_AND_CYTOPHAGA 7.1% 7.1%CHLOROFLEXUS_SUBDIVISION 3.6% 3.6% CY.AURANTIACA_GROUP 7.1% 7.1%CYANOBACTERIA 7.1% 7.1% DELTA_SUBDIVISION 14.3% 14.3%ENVIRONMENTAL_CLONE_WCHB1- 7.1% 7.1% 41_SUBGROUP FLX.LITORALIS_GROUP3.6% 3.6% GAMMA_SUBDIVISION 10.7% 10.7% HIGH_G + C_BACTERIA 7.1% 7.1%LEPTOSPIRILLUM GROUP 3.6% 3.6% MYCOPLASMA_ AND_RELATIVES 3.6% 3.6%PIRELLULA_GROUP 3.6% 3.6% SPHINGOBACTERIUM_GROUP 3.6% 3.6%SPIROCHAETA-TREPONEMA- 3.6% 3.6% BORRELIA_SUBDIVISIONTHERMOANAEROBACTER_AND_RELATIVES 3.6% 3.6%

Example 3 Multitag Pyrosequence Analysis of Dysbiosis in IBP

Inflammatory Bowel Diseases (IBD or IBDs), namely ulcerative colitis(UC) and Crohn's disease (CD), are chronic, lifelong, relapsingillnesses, affecting close to 1 million Americans and costingapproximately $2 billion per year to the US healthcare system. IBDs areof unknown cause, have no cure, and are increasing in incidence. Thenatural course of these diseases is characterized by periods ofquiescence (inactive disease) interspersed with flare-ups (activedisease). It is now widely accepted that flare-ups of IBD are due to adysregulated inflammatory reaction to abnormal intestinal microfloradysbiosis), however.

Specific changes in the microflora of IBD patients that might causethese diseases remain unknown. Narrow searches for a single pathogenthat causes IBD have been unsuccessful. (See Guarner and Malagelada2003). Studies of small bacterial groups have yielded ambiguous results.(See Schultz and Sartor 2000). Only recently have studies of large setsof bacterial flora been attempted. (See Eckburg, Bik, et al. 2005).Improving our knowledge about GI tract microflora has the potential torevolutionize IBD treatment. Development of real-time methods to studymicrofloral changes may lead to diagnostic tools to predict flare-ups,and to targeted, safe treatments for IBD.

The key requirement to understanding dysbiosis in polymicrobial diseasesis for a method to interrogate widely the microflora in numerous controland disease samples to identify dynamic trends in species compositionassociated with health and disease progression. In classic communitystudies (Eckburg, Bik, et al. 2005) environmental samples are assayedindependently and then the clone sequence data from specificclasses/groups are statistically analyzed usually using some sort ofaveraging metric. This can be extremely costly, especially if the clonelibraries are exhaustively analyzed (i.e., 10,000 clones per sample).

To improve throughput and reduce cost, Amplicon Length Heterogeneity PCR(ALH-PCR) has been used to study the gut microflora. It offers a rapidway of screening complex microbial communities, allowing for easyfingerprinting of microfloral changes. The LH-PCR fingerprinting isinexpensive and fast, with the ability to screen several hundred samplesa day. It can be used as a routine survey tool to monitor the dynamicsof natural soil microbial communities or to quickly identify samples ofinterest using PCO analysis. PCO analysis has been used to group sampleswith similar profiles, allowing them to be pooled for cloning andsequencing. This greatly reduces the cost of analyzing multiple samples,particularly when the analysis requires sequencing at least 300 randomclones to identify bacterial components of the sample down to 1%representation in the total population (which is the resolution limitfor ALH fingerprinting). Pooling similar samples before cloning andsequencing has proved to be quite robust. However, equal amounts of thePCR product front each sample must be pooled or the results will beskewed.

Multitag Pyrosequencing is a novel pyrosequencing technology that allowsmany community samples to be sequenced together at high coverage withoutthe necessity for fingerprinting, cloning, or the purification andseparation techniques required by conventional methods for analyzingmicrobial communities, as described herein above. Multitag sequencing ismore efficient, faster, and less costly than other methods.

By way of illustration, Multitag Pyrosequencing can be carried out usinga set of specific tags on the end of standard universal small ribosomalsub-unit (“SSU”) rRNA primers (See Table 1). A different set of thetagged primers is used to amplify the SSU rRNA in each differentenvironmental sample (FIG. 2—Step 1). The PCR amplicons from all thesamples are pooled. Emulsion PCR is performed and the amplicons arisingfrom each molecule are captured on their respective beads. Followingamplification, the beads are distributed into the wells of a picoliterplate (FIG. 2—Step 2). The sequences, including the tagged sequences, ofthe amplicons on each bead are determined by pyrosequencing (FIG. 2—Step3). A PERL script or other suitable program is used to sort the sequenceinformation using the tags and primer sequence as a key. Sequences withthe same tags are identified thereby with their respective sample. Thebacteria species in each sample then are identified by matching the SSUrRNA sequences to entries in the database of the Ribosomal DatabaseProject (either RDP 8.1 or RDP 9.0). The normalized frequency with whicha bacteria is thus identified in a given sample is indicative of itsrelative representation in the microbial community. Histograms based onthese frequency determinations can be used for the non-parametricanalysis of dysbiotic shifts involved in disease states.

For example, FIG. 3 depicts the results of such an experiment in whichsix Control, ten Crohns, and eight Ulcerative colitis mucosal sampleswere analyzed by Multitag Pyrosequencing. Each of the segments in thestacked histogram bars represents the normalized abundance of thatspecific taxa in a specific sample. In this experiment, identificationof the taxa was performed using BLAST analysis of the RDP 8.1 database.It can be seen that some taxa (i.e. Bacillus fragilis subgroup andRumanococcus gnavus subgroup) are present in the same abundance in bothcontrol and disease states. Other taxa, such as Clostridium leptum aremore dominant in Ulcerative colitis, while others (i.e. the Gloeothecegloeocapsa subgroup) are indicators of dysbiosis in the disease state.

However, the standard 454 Life Science process using a ligation step tolink the emulsion FOR adapters to the PCR amplicons and producesnumerous artifacts in the quantitation of the abundances of each taxa inthe samples. In the results displayed in FIG. 3, we algorithmicallyremoved chimeras, reverse reads and truncated products and filtered thedata to remove all taxa that were represented by less than 5% abundance.Only then were we able to see a correlation with disease state andspecific microbial taxa.

Example 4 Distortion of the Distribution of Components of a MicrobialCommunity by Directly Ligating Emulsion PCR Adapters onto PCR Amplicons

In one experiment we used tagged PCR primers to amplify the componentsin duplicate microbial community samples, ligated the Emulsion PCRadapters to these samples, and then subjected these samples to separatepyrosequencing runs. The amplicons are routinely run on an AgilentBioanalyzer system before and after ligation to quantitate the mixturebefore emulsion PCR. FIG. 4 depicts a sample run on the Bioanalyzerbefore and after direct ligation and clearly shows that the ligationstep has drastically altered the distribution of the amplicons.

Additionally, we compared the normalized abundances of the componenttaxa identified by the multitag process after direct ligation of theEmulsion PCR adapters. In this experiment, identification of the taxawas performed using a Bayesian analysis of the RDP 9.0 database. We cansee in FIG. 5 that abundances of the forward and reverse primers forvarious taxa are different within a sample and between duplicatesamples. In several cases, we are missing entire families in thecomparison between duplicates. Table 3 summarizes the differencesbetween the forward primers and the reverse primers of the duplicatesamples and it is clearly stochastic with no predictable pattern. Wehypothesize that this differential ligation efficiency could be due to anumber of factors such as internal structure in the amplicons or biasesin the terminal nucleotide of either the adapter or amplicon.

TABLE 3 Duplicate Sample Analysis FORWARD REVERSE PRIMERS PRIMER RDP 9.0FAMILY RATIOS RATIOS Acidaminococcaceae 544.6% 195.0% Actinomycetales144.0% 116.5% Bacteroidaceae 119.9% 124.5% Clostridiaceae 97.5% 99.4%Comamonadaceae 198.0% Coriobacteriales 181.5% 141.5% Enterobacteriaceae4.2% Eubacteriaceae 88.0% 87.5% Flavobacteriaceae 34.9% Incertae sedis 9106.4% 143.0% Lachnospiraceae 176.8% 113.1% Peptococcaceae 91.0%Peptostreptococcaceae 94.7% 115.4% Porphyromonadaceae 99.0% 97.3%Prevotellaceae 264.0% 88.1% Rikenellaceae 212.2% 106.1% Streptococcaceae74.3% 60.7%

LITERATURE CITED

Each of the following publications is incorporated herein by referencein its entirety, particularly as to the above-referenced subject matter,especially relating to methods that can be employed in carrying outmultitag sequencing and/or relating to uses thereof.

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1.-26. (canceled)
 27. A kit comprising at least five forward and reverseprimer pairs, wherein each forward and reverse primer comprises, in 5′to 3′ order: a priming sequence, a tag sequence of from 4 to 36nucleotides in length, and a probe sequence targeting a genetic regionfor amplification, wherein: the priming sequence is the same between theprimer pairs, with the proviso that forward and reverse primers may havethe same or different priming sequences; the tag sequences in eachprimer pair are unique so as to identify the primer pair used in anamplification reaction, wherein the tag sequences differ by at least twonucleotides; and the probe sequences between the primer pairs are thesame, and hybridize 3′ to a variable genetic region selected from: a 16SrRNA variable sequence, an 18S rRNA variable sequence, and an ITSvariable sequence, so as to amplify a population of variable sequencesin a sample.
 28. The kit of claim 27, wherein the genetic region variesbetween species.
 29. The kit of claim 27, wherein the genetic regionvaries within a species.
 30. The kit of claim 27, further comprising aprimer complementary to and effective for elongation from the primingsequence.
 31. The kit of claim 27, wherein the kit comprises from 10 to25 primer pairs.
 32. The kit of claim 27, wherein the kit comprises from20 to 50 primer pairs.
 33. The kit of claim 27, wherein the kitcomprises from 50 to 150 primers pairs.
 34. The kit of claim 27, whereinthe kit comprises from 100 to 500 primers pairs.
 35. The kit of claim27, wherein the tag sequences are 4 nucleotides in length.
 36. The kitof claim 27, wherein the tag sequences are 5, 6, 7, 8, 9, 10, 11, or 12nucleotides in length.
 37. The kit of claim 36, wherein the tagsequences are 6 nucleotides in length.
 38. The kit of claim 36, whereinthe tag sequences are 7 nucleotides in length.
 39. The kit of claim 36,wherein the tag sequences are 8 nucleotides in length.
 40. The kit ofclaim 27, wherein each of the primer pairs is disposed separately fromone another in a microtiter plate.
 41. The kit of claim 27, wherein thetag sequences in a primer pair are the same.